Emergence of the invasive Asian bush mosquito Aedes (Hulecoeteomyia) japonicus (Theobald, 1901) in the Czech Republic

Background Aedes japonicus is a mosquito species native to North-East Asia that was first found established outside its original geographic distribution range in 1998 and has since spread massively through North America and Europe. In the Czech Republic, the species was not reported before 2021. Methods Aedes invasive mosquitoes (AIM) are routinely surveyed in the Czech Republic by ovitrapping at potential entry ports. This surveillance is supported by appeals to the population to report uncommon mosquitoes. The submission of an Ae. japonicus specimen by a citizen in 2021 was followed by local search for aquatic mosquito stages in the submitter’s garden and short-term adult monitoring with encephalitis virus surveillance (EVS) traps in its surroundings. Collected Ae. japonicus specimens were subjected to nad4 haplotype and microsatellite analyses. Results Aedes japonicus was detected for the first time in the Czech Republic in 2021. Aquatic stages and adults were collected in Prachatice, close to the Czech-German border, and eggs in Mikulov, on the Czech-Austrian border. Morphological identification was confirmed by molecular taxonomy. Genetic analysis of specimens and comparison of genetic data with those of other European populations, particularly from Germany, showed the Prachatice specimens to be most closely related to a German population. The Mikulov specimens were more distantly related to those, with no close relatives identifiable. Conclusions Aedes japonicus is already widely distributed in Germany and Austria, two countries neighbouring the Czech Republic, and continues to spread rapidly in Central Europe. It must therefore be assumed that the species is already present at more than the two described localities in the Czech Republic and will further spread in this country. These findings highlight the need for more comprehensive AIM surveillance in the Czech Republic. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05332-5.


Background
The presence of four Aedes invasive mosquito (AIM) species in Europe is consistently noted: Aedes aegypti, Aedes albopictus, Aedes japonicus and Aedes koreicus have been introduced into many European countries and have succeeded in establishing in several of them. Rather than being a nuisance, these mosquito species may pose a serious threat to public health by their capability of transmitting arboviruses such as dengue, chikungunya, yellow fever and Zika viruses. The intercontinental spread of these AIMs is facilitated by global trade [1] and is attributable to their drought-resistant eggs [2]. Establishment and further dispersal in the new geographical regions are possible when climatic and environmental conditions are adequate. In Central Europe, Ae. japonicus and Ae. koreicus find similar (moderate) climatic conditions as in their

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Parasites & Vectors *Correspondence: helge.kampen@fli.de; helge.kampen@fli.de original Asian distribution range, while the thermophilic species Ae. albopictus increasingly adapts to moderate temperatures and takes advantage of climate warming.
Aedes (Hulecoeteomyia) japonicus (Theobald 1901), also known as the Japanese bush mosquito or Asian rock pool mosquito, is native to North-East Asia [3]. Its first finding in Europe was reported in 2000 from north-western France, where two larvae were collected in car tyres [4]. Subsequently, the species was found in Belgium in 2002 as larvae and in 2004 as adults [5]. Later reports describe occurrences in Switzerland and Germany in 2008, Austria and Slovenia in 2011, and the Netherlands and Hungary in 2012 [6][7][8]. In 2013, the establishment of Ae. japonicus was confirmed in France [9], and in the same year, the emergence of this mosquito was reported from Croatia [10]. Its spread continued in 2015 to Liechtenstein and Italy, in 2017 to Bosnia and Herzegovina, and in 2018 to Serbia, Spain and Luxembourg [11][12][13][14]. The most recent report of Ae. japonicus introduction was added from Romania for 2020 [15].
Unlike Ae. albopictus, which is exclusively spread by human mediation, Ae. japonicus spreads to new areas by active dispersal, too [16,17], with spreading corridors mainly being the borders between rural and urban habitats, such as suburban gardens, vineyards, small patches of forest adjacent to fields or forest edges with streams [12,18,19]. Adults usually live in forests but migrate to gardens or cemeteries to lay eggs, where they find more potential breeding sites, i.e. artificial containers with stagnant water [20]. In the United States, larvae of Ae. japonicus are commonly found in rock pools, similar to their native range, but in Europe they have also been collected from tree holes and a variety of man-made containers such as barrels, tyres, bathtubs or flower vases [12,13,21]. Aedes japonicus typically seeks larger containers than other container-breeding AIMs [19,21]. In Switzerland and Germany, the species Aedes geniculatus and Anopheles plumbeus use similar breeding sites as Ae. japonicus [18,20].
Moreover, Ae. japonicus is well adapted to cold winters and snow. Its larvae have been found in water as cold as 4 °C [16]. Its occurrence above 700 m above sea level (a.s.l.) in south-eastern Europe [12] and at 1200 m a.s.l. in the German Black Forest [22] suggests that crossing mountains should not be very problematic for this species. In the southern Appalachians in the United States, Ae. japonicus was the only container-breeding mosquito species found above 1400 m a.s.l. [21].
Aedes japonicus feeds on a variety of hosts, but appears to be less anthropophilic than other AIMs [23].
There is no evidence of pathogen transmission by Ae. japonicus under natural conditions. Therefore, this species is considered less important as a public health vector than Ae. aegypti or Ae. albopictus. However, genetic material of West Nile virus (WNV) has been demonstrated several times in Ae. japonicus collected in the field in the USA [24,25]. Furthermore, in laboratory studies, this mosquito shows vector competence for eastern equine encephalitis, La Crosse, St. Louis encephalitis, Rift Valley fever, Usutu, chikungunya, dengue and Zika viruses [26][27][28][29][30][31] in addition to WNV [25], as well as for the nematodes Dirofilaria repens and Dirofilaria immitis [32].
We here describe the first encounters with Ae. japonicus mosquitoes in the Czech Republic, which took place in 2021, independently in two areas more than 190 km apart. One finding locality was localised close to the border with Germany and the second on the border with Austria.

Study sites and mosquito collection
In the evening hours of 15 June 2021, a citizen of the town of Prachatice in South Bohemia caught a strangely coloured adult female mosquito in his house. He sent five pictures of this mosquito to our laboratory for preliminary identification. Since it was not possible on these to distinguish between Ae. japonicus and Ae. koreicus, the pictures were followed by the physical specimen for accurate identification. On 9 July, two additional adult mosquitoes of the same appearance (one male, one female) were captured by the citizen at the same location.
From 11 to 13 August, the garden of the reporting resident and its surroundings were checked for AIMs by dipping water containers for aquatic mosquito stages and trapping adult mosquitoes by encephalitis virus surveillance (EVS) traps equipped with dry ice as an attractant. Sampling of aquatic mosquito stages was done repeatedly on 11 and 12 August from three dark green plastic rainwater collection barrels, that stood immediately against the house wall, each holding approximately 300-400 L of water and having a water surface area of about 0.8 m 2 . From 11 to 12 August, eight EVS traps were operated within 100 m and another seven traps within 1.5 km from the submitter's house. Two EVS traps were placed even further away (about 3.5 km). The traps were located at shady places near the gardens of family houses, in an allotment area and on the edge of a forest. Three of the traps in the 100 m diameter were placed about 20 m from the rainwater barrels, another two about 50 m and three about 80 m. One of the three traps within 20 m distance was equipped with sweaty socks as an additional attractant. All traps were run overnight. On 12 August, one trap was kept working all day next to the rainwater barrels. From the evening of 12 August to the morning of 13 August, a total of 14 EVS traps were operated in close vicinity (100 m) of the rainwater barrels in the submitter's garden.
The city of Prachatice ( Fig. 1) is situated in the foothills of the Bohemian Forest and borders its protected landscape area, which includes the Bohemian Forest National Park. The town is located in a valley basin with a flowing stream at an altitude of 561 m a.s.l. In the surrounding area, there are many suburbs with gardens and gardening colonies. The landscape is hilly and consists of forest and meadows rather than agricultural land. About 25 km to the south-east runs the German-Czech border, with Ae. japonicus occurrence reported not far from the border on the German side (Kampen & Werner, unpublished data).
Ovitrapping was done in the framework of a long-term surveillance programme focusing on the occurrence of Ae. albopictus at the border between the Czech Republic and Austria [33,34]. The site 'Mikulov 2' (Fig. 1) is one of three parking lots in the outskirts of the town of Mikulov included in the monitoring programme. This place is located between the motorway A5 on the Austrian side and the motorway 52 to Brno on the Czech side and is used by lorry drivers as a stopover. For a detailed description of the surveillance methodology by ovitrapping, see Rudolf et al. [34].

Rearing and identification of mosquitoes
Oviposition supports with eggs from ovitraps were brought to the laboratory where the eggs were flooded in jars with stale tap water for the larvae to hatch. Hatched larvae were fed ground fish food (Tetra GmbH, Melle, Germany). Larvae from the rainwater barrels were transferred to jars together with original barrel water, the organic compounds of which served as larval food during further development. Jars were covered with nets until emergence of adults, which were collected with a battery-powered aspirator (Hausherr's Machine Works, Toms River, NJ, USA) and killed by freezing at −20 °C for at least 24 h.
Adults were morphologically identified using the determination key provided in Becker et al. [35]. Morphological determination was confirmed genetically on one specimen from Prachatice and two specimens from Mikulov (adults). For this purpose, one leg per mosquito specimen was individually homogenised in 150 μl sterile demineralised water with sterile 5 mm steel beads at 30 Hz for 60 s in a TissueLyser II (Qiagen, Hilden, Germany). Genomic DNA was extracted from Fig. 1 Location of Ae. japonicus collection sites in the Czech Republic (nos. 1, 2) and of Ae. japonicus populations genetically compared with them (nos. [3][4][5][6][7][8][9]. Names of locations are provided in Table 1; colours of dots refer to the two genetic clusters as shown in Fig. 3 100 μl of the homogenates using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. The DNA was eluted in 80 µl elution buffer and stored at −20 °C until further processing.

Population genetic analysis
To obtain clues on the origin of the Czech Ae. japonicus samples and their relationships to conspecific populations in Europe, population genetic analyses were performed on 20 individuals from Prachatice, 14 individuals from Mikulov and 20 individuals from Grafenau, the closest German place to the Czech border and Prachatice known to be colonised by Ae. japonicus (beeline distance between Grafenau and Prachatice ca. 50 km). The data obtained from these three locations were compared with data from previously investigated Ae. japonicus populations from other areas in Germany and one location in Croatia (Table 1). Analysis was performed on nad4 haplotypes and microsatellites. For the latter, seven polymorphic loci (OJ5, OJ10, OJ70, OJ85, OJ100, OJ187, OJ338) were genotyped as described by Janssen et al. (2019). The results of the microsatellite analysis were interpreted with Geneious Prime version 2021.0.1 (Geneious Biomatters, Auckland, New Zealand), subjected to a Bayesian cluster analysis using STRU CTU RE [41] and evaluated with STRU CTU RE HARVESTER [42]. Furthermore, based on Nei's genetic distance and pairwise F ST values, a principal coordinate analysis (PCoA) was performed.

Mosquito collection and identification
The first adult female mosquito from the Prachatice locality was identified as Ae. japonicus on the basis of characteristic morphological features-scutal stripes, scales on the sides of the thorax, tergite scale pattern and hind leg colouration (Fig. 2). Molecular analysis of that specimen revealed 99.86% similarity of the cox1 partial sequence (GenBank accession no. OM307664) to Ae. japonicus GenBank accession no. KF211505 from Germany and 99.35% similarity of the obtained nad4 partial sequence (GenBank accession no. OM307666) to Ae. japonicus GenBank entry AF305879 from Germany.
Adults developed from several dozen larvae and pupae collected from the three rainwater barrels (out of hundreds present) were also morphologically identified as Ae. japonicus. Other mosquito species detected in the barrels belonged to the Culex pipiens and Anopheles maculipennis complexes.
No Ae. japonicus was collected in the EVS traps during the first night of trapping in Prachatice, but one Ae. japonicus female was captured during the second night with the EVS trap equipped with sweaty socks. All other EVS traps remained negative for Ae. japonicus. However, Cx. pipiens complex females and some Aedes vexans specimens were collected by the EVS traps within the 100 m radius, and several individuals of Ae. geniculatus and An. plumbeus were collected from EVS traps placed on the forest edge. One EVS trap placed in a garden colony contained an An. maculipennis complex female.
Thirty-six morphologically identified adult Ae. japonicus (25 females, 11 males) emerged between 28 and 30 August 2021 from ovitrap egg collections carried out from 10 to 18 August 2021 at 'Mikulov 2' . The sequences of the two tested Ae. japonicus individuals were  [40] identical (GenBank accession nos. OM307665 for cox1 and OM307667 for nad4) and shared 99.58% nucleotide homology with a cox1 sequence from an isolate from Germany (GenBank accession number KF211505) and 99.14% nucleotide homology with a nad4 sequence from an isolate from the USA (GenBank accession number AF305879).

Population genetic analysis
Nad4 haplotype analysis was performed on 54 specimens from Prachatice, Mikulov and Grafenau (south-eastern Germany). The alignment indicated 21 variable nucleotide positions, leading to four different nad4 haplotypes: H1, H3, H9 and H21 (Table 2). Six individuals were characterised by heteroplasmy, the presence of different mitochondrial DNA (mtDNA) variants in one organism, as indicated by peaks for two different nucleotides at the same position in the sequencing electropherogram. Analysable microsatellite data were obtained for 53 samples from the two Czech populations and the German population from Grafenau. For comparison, data available from previous studies on German and Croatian Ae. japonicus populations were included in the analysis (Additional file 1: Table S1). The Bayesian cluster analysis showed the highest probability for the existence of two genetic clusters among the tested populations (k = 2; ∆ = 160.08). According to this analysis, the populations  from Prachatice, Mikulov, Grafenau and Macelj (northwestern Croatia) have a high probability of belonging to the same microsatellite genetic cluster 1 (blue colour), whereas the previously investigated populations from Germany rather appear to belong to genetic cluster 2 (red colour) (Fig. 3). Despite the same principal genetic cluster 1, the Mikulov population seems to be somewhat different from the Prachatice, Grafenau and Macelj populations.
The results of the PCoA, based on F ST values and Nei's genetic distance of the microsatellite locus data, show a close genetic relatedness of the Prachatice and Grafenau populations. The other included populations are much more distantly related (Fig. 4).

Emergence of Ae. japonicus in the Czech Republic
The Bohemian Forest region in which the town of Prachatice is embedded provides appropriate landscape structures and paths for the spread of Ae. japonicus (e.g. more rural than urban area, presence of many deciduous forest patches and occurrence of transition zones between forest and local settlements) [18,20]. Based on the demonstrated presence in close-by Germany and the genetic data, an origin of the Ae. japonicus specimens from Prachatice and a (possibly active) spread of Ae. japonicus from Germany can be assumed. This, however, suggests that Ae. japonicus is likely to have already been established between Prachatice and the Czech-German border as well and would explain the high abundance of larvae (hundreds) found in the rainwater barrels in the submitter's garden, which were hardly produced by a single female. Amazingly, no adult specimens were trapped in Prachatice but this could be due to poor trappability of this species by EVS traps [43]. Larval sampling in Prachatice and the area west of it is planned for 2022 to check for further Ae. japonicus occurrence.
Locality 'Mikulov 2' is a parking lot with the first gas station on the Czech side of the Czech-Austrian border.  'Mikulov 2' is attributable to passive transport and introduction via long-distance traffic of a single gravid female along the highway from the south or to active dispersal. The latter is assumed to have taken place with specimens of Ae. japonicus found in the framework of the Austrian mosquito monitoring programme in the Lower Austrian district of Gmünd [44], about 10 km from the Czech border and far from an international traffic route.

Population genetic analysis
The most frequent nad4 haplotype found in the Ae. japonicus populations from Prachatice, Mikulov and Grafenau was H9. This nad4 haplotype is known from several populations in Europe: Belgium, Austria, The Netherlands, Slovenia and Croatia [12,39,40,[45][46][47]. Nad4 haplotype H3 was exclusively detected in Prachatice. This haplotype has been found in populations in South Germany, The Netherlands and Bosnia and Herzegovina [12,45]. A carry-over by mosquito displacement from those populations to the Czech Republic is possible. The same is true for nad4 haplotype H21, as found in both Czech populations in this study, which had previously been detected in South Germany and Bosnia and Herzegovina [12,45]. In summary, since Prachatice is represented by all four haplotypes found in this study and both Mikulov and Grafenau by two each, but different ones (Table 2), the haplotype analysis is not informative enough to deduce relatedness of the tested populations and displacement/migration routes.
With regard to the results of the microsatellite analysis and the PCoA (Figs. 3, 4), the spatially close collection sites Prachatice and Grafenau show high genetic relatedness, with the second next closest relationship to the populations from Macelj (microsatellite multilocus genotype) or Augsburg and Berchtesgaden (PCoA). A common origin of these populations could be in Austria or Slovenia, where Ae. japonicus is widely distributed [48]. The Mikulov population seems to be genetically more different from the other populations tested. A new introduction from a European population not included in the analysis or even from overseas could be the reason.

Conclusions
Invasive Ae. japonicus mosquitoes were reported for the first time in the Czech Republic. Due to the low diversity of nad4 haplotypes, the two populations from the Czech Republic as well as that from Grafenau, south-eastern Germany, can be assumed to be relatively young. However, owing to the already wide distribution of Ae. japonicus in Europe with ongoing mixture of populations, haplotypes cannot be assigned to certain populations anymore as was the case during the first years of invasion of the western world (e.g. [38,49,50]). According to microsatellite and PCoA analyses, the Prachatice population was most likely introduced to the Czech Republic from Germany, while the Mikulov population is at least admixed with genetic material from one (or several) other population(s) of unknown origin.